The present invention relates generally to high resolution, three dimensional, fluorescence microscopy systems and methods for their use. More, specifically the invention represents specific improvements to existing wide-field, optical sectioning microscopes systems designed for acquisition and analysis of multi-dimensional fluorescence images.
The field of optical microscopy has been revolutionized in recent years by the widespread use of confocal and fluorescent microscopes. Combining laser illumination and digital image processing, these optical instruments allow biologists to obtain high-resolution, three dimensional fluorescent images.
Deconvolution microscopy, an alternative approach to laser-scanning cofocal microscopy, is gaining in popularity. This technology avoids the high costs and limitations of laser illumination and is ideal for live-cell studies requiring high resolution and multiple wave lengths. Typical of the devices of this type is the Delta Vision(copyright) microscope system. Delta Vision(copyright) system is a wide-field optical sectioning microscope system. See, for example, info@api.com and U.S. Pat. No. 5,684,628. The basic concepts embodied in the Delta Vision(copyright) system include: collection of imaging data directly in digital form using a high quality charge coupled device, i.e., CCD (silicon chip) area detector; computation removal of noise arising from light leakage into the focal plane from adjacent planes, provision of a facile user interface for data capture and analysis.
The invention is directed to improvements to a standard wide field microscope system, e.g. the Delta Vision(copyright) system. The improvements permit imaging of more than 1,000, preferably more than 10,000 samples, e.g. antibody/antigen reactions, per day. The basic system to which these improvements are applied share generally the features of a fluorescent microscopexe2x80x94i.e., excitation and emission filters, an objective lens, a movable stage, and image recording by CCD. Such basic instruments also are able to image transmitted light.
The features which constitute improvements to this basic design fall into three general categories:
Optical features which constitute the improvements of the invention include a pulsed light source, a fiber-optic light source, a computer-controlled condenser, infinity focusing, polychromatic beam splitting and multiple CCD detectors rather than a single CCD detector.
Mechanical features which comprise the improvements of the invention include a rotatable circular disc in place of a conventional sample holder, wherein the disc may also contain further design features creating separate small wells; reagent dispenser and readout stations positioned around the disc, an arm that moves the dispenser and readout heads radially; a stage-tilting device, and a temperature-controlled specimen chamber.
In addition, the improvements comprise computational features including a control and calibration program, a nonhomogeneous illumination compensator, chromatic aberration compensator, a Fourier space feature detector, and a real space feature detector.
One object of the innovation is to minimize photodamage to the samples by pulsing the light source at a speed consistent with the rate of data collection. Fiber optics plumbing, pre- and post-specimen, with a rotating dichroic filter in the illumination path, enables more efficient automated operation. An automated condenser (e.g. to slide in masks for differential interference and polarization imaging) is also advantageous.
Another aspect of the invention relates to infinity imaging. This employs an objective lens with a sliding element to allow for switching between high and low magnification, a more efficient system than conventional rotating head microscopes. The image is further focused by sliding a secondary lens into the light path downstream from the objective lens. This simplifies positioning of the objective lens since the computer controlled motor for focusing can be placed physically out of the way. xe2x80x9cInfinity imagingxe2x80x9d also enhances the range of magnification possibilities, and eliminates moving parts from the proximity of the sample. This simplifies temperature control, for example permitting cooling of the specimen. Cooling the specimen sharpens the emission line widths enhancing the ability to recognize differences in hues.
A still further aspect of the invention is the use of beam splitters in conjunction with filters to allow the same primary light to be imaged onto multiple CCD detectors. Multiple detectors allow faster collection of data and more reliable image registration for detection of multiple colors.
In mechanical features, in one aspect, the invention employs rotatable circular discs in place of the typical microscope slides or 96-well plates. These discs are placed on a rotatable stage. Typically, the disc can range in size from a 4-inch diameter to 9 or 12 inches. The exact size of the disc is not critical. These discs, which may be made of glass, are also available from the computer chip industry. The discs can be painted with hydrophobic materials to form barriers which would define sample wells using lithographic techniques. The lithographic techniques employed are similar to those taught in U.S. Pat. No. 5,212,028, the contents of which are incorporated herein by reference. Using these techniques, a 4-inch disc can be painted, for example, to form 2 mm segments which creates 1,000 sample wells and to form 1 mm segments which creates 2,500 wells. Alternatively, adhesive lines that allow the deposit of small specimen carriers can be used.
Other advantageous mechanical features of the invention are related to the use of these multispecimen discs. Thus, reagent dispensers and readout stations may be positioned around the disc and may be fitted with arms that move the dispensers and read-heads radially. Other mechanical features include a stage-tilting device and a temperature controlled specimen chamber, facilitated by the infinity focusing feature.
Computational aspects of the invention include dedicated circuitry and equivalent software for computational processing in areas which include data capture, photon reassignment feature detection, counting and presentation. These measures facilitate control and coordinate data handling. Further, the circuitry and software equivalent permit the recognition of signature curves of particulate labels such as latex beads. This would permit the system to count and identify individual tags. An alternative filter can be designed to read microbar codes for sample tracking.
Other filters can also be defined computationally to improve the accuracy of cell counting. Cell borders can be recognized by virtue of lipid soluble dye, while definition of the nucleus can be achieved using a DNA stain like DAPI.